Application of BCAM in preparation of POP diagnosis and evaluation product and product

Abstract

The invention discloses application of BCAM in preparation of a POP diagnosis and evaluation product and the product, and belongs to the technical field of biological medicine, and the product is a substance which takes a blood sample of a subject as a detection object and is used for detecting the BCAM level in the blood sample. The expression level of the BCAM protein in blood is taken as a detection standard, the evaluation condition that whether a subject suffers from POP or not can be accurately and specifically reflected, and a new detection index is provided as guidance and basis for clinical diagnosis and evaluation of POP.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of BCAM in the preparation of POP diagnostic and evaluation products and related products. Background Technology

[0002] Pelvic organ prolapse (POP) is a common condition in middle-aged and elderly women, caused by weakened pelvic floor muscles and fascia, leading to the descent and dysfunction of pelvic organs. Main symptoms include anterior vaginal wall prolapse, posterior vaginal wall prolapse, uterine prolapse, and vaginal fornix prolapse, resulting in the herniation of adjacent organs into the vaginal area, i.e., cystocele, rectocele, and enterocele in the traditional sense. The main manifestations are the prolapse of tissue at the vaginal opening and a feeling of heaviness or pressure, which may be accompanied by urinary, bowel, and sexual dysfunction, affecting the patient's quality of life to varying degrees.

[0003] Currently, the diagnosis and assessment of POP (post-oral ovarian prolapse) are mainly differentiated through clinical phenotype, medical history, and relevant functional examinations. Typically, severely affected patients may experience vaginal prolapse, or varying degrees of lumbosacral pain or a feeling of heaviness, making diagnosis easily achievable during clinical examination. However, mild cases generally present without discomfort, requiring doctors to make a diagnosis based on different functional examination results and their experience, which can easily lead to misdiagnosis or missed diagnosis. Therefore, exploring and researching relevant biomarkers is of great significance for the diagnosis and prevention of POP.

[0004] In the prior art, Chinese patents CN111856038A, CN111596069A, CN111596068A, and CN111596067A disclose UNC45A (Unc-45 myosin chaperone protein), HSP10 (heat shock protein 10), Utrophin (a protein associated with Duchenne muscular dystrophy), and ZC3H8 (CCCH type zinc finger protein), which can be used as relevant detection indicators for the diagnosis of POP. By comparing the detection indicators with the standard values ​​of normal individuals, if there are abnormalities, POP can be diagnosed. These patents play a very important role in the clinical diagnosis of POP.

[0005] BCAM protein is a member of the immunoglobulin superfamily and a receptor for the extracellular matrix protein laminin. Existing research indicates that BCAM can inhibit trophoblast proliferation, migration, and invasion by suppressing PIK3R6 / p-STAT3 signaling, potentially leading to placental dysfunction and a preeclampsia-like phenotype. BCAM also has a role in promoting ovarian cancer metastasis. However, there are currently no reports on the use of BCAM in the diagnosis and evaluation of pompholyx (POP). Summary of the Invention

[0006] The purpose of this invention is to provide the application and products of BCAM in the preparation of POP diagnostic and assessment products. Using the expression level of BCAM protein in blood as the detection standard, it can accurately and specifically reflect the assessment of whether the subject has POP, and provides a new detection indicator as guidance and basis for the clinical diagnosis and assessment of POP.

[0007] This invention is achieved through the following technical solution: the application of BCAM in the preparation of POP diagnostic and assessment products, wherein the products are substances that use blood samples of subjects as the detection object and are used to detect the level of BCAM in the blood samples.

[0008] The blood sample may be whole blood, serum, or plasma.

[0009] The substance in question is used to detect the level of BCAM at the protein level.

[0010] The detection methods include immunofluorescence assay, ELISA, biochip assay, and electrochemiluminescence assay.

[0011] The products include reagent kits, test strips, or other medically feasible products.

[0012] Furthermore, the present invention also provides a POP diagnostic and assessment product for use in the above-described applications.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention provides a new detection index for the diagnosis and assessment of POP. By detecting the expression level of BCAM protein in blood samples as a basis for judgment, it can provide a rapid identification method for the clinical diagnosis and assessment of POP. Experiments have shown that the expression of BCAM protein can accurately and specifically achieve the clinical diagnosis of POP. Therefore, it can provide a new detection basis and standard for the clinical diagnosis and assessment of POP.

[0014] (2) When performing BCAM protein level expression detection, the present invention can directly use known methods such as immunofluorescence analysis, ELISA, biochip method, electrochemiluminescence method, etc. to directly obtain detection results, and can achieve high sensitivity, accurate and rapid detection. Attached Figure Description

[0015] Figure 1 This is the A450 value-BCAM concentration standard curve involved in Example 2. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0017] Example 1: Preparation of a POP diagnostic and evaluation kit for BCAM detection Reagent preparation: Carbonate buffer at pH 9; Carbonate buffer at pH 9.6 with a concentration of 1 mol / L; Anhydrous methanol; Bovine serum albumin solution; Phosphate buffer at pH 7.2 with a concentration of 0.05 mol / L; Dimethylformamide; Biotinyl-N-hydroxysuccinimide; Avidin-horseradish peroxidase; 3',3',5,5'-Tetramethylbenzidine; BCAM protein standard; Sample diluent.

[0018] Instrument preparation: constant temperature chamber; magnetic stirrer; precision pipette; microplate reader capable of measuring absorbance at 450nm.

[0019] The specific preparation steps are as follows: (1) Prepare multi-well plates coated with mouse anti-human BCAM monoclonal antibody (purchased from Novus, catalog number H00000608-M06).

[0020] The mouse anti-human BCAM monoclonal antibody was diluted to a concentration of 10 μg / mL using carbonate buffer at pH 9. Anhydrous methanol was added to the antibody dilution, with the amount of anhydrous methanol limited to a concentration of 3 mL / 100 mL. Then, 100 μL of the methanol-containing mouse anti-human BCAM monoclonal antibody dilution was added to each well of a 96-well plate, and the plate was coated at 4°C for 12 hours. After the coating time was up, 120 μL of 1 g / 100 mL bovine serum albumin solution was added to each well of the multi-well plate, and the plate was blocked at 37°C for 1 hour. After the blocking reaction, the multi-well plate was washed with phosphate buffer at pH 7.2 and a concentration of 0.05 mol / L. Once the unreacted material on the multi-well plate was removed, the multi-well plate coated with mouse anti-human BCAM monoclonal antibody was obtained and stored at 4°C for later use.

[0021] (2) Preparation of biotin-labeled rabbit anti-human BCAM polyclonal antibody (purchased from Abcam, catalog number ab134110) (Biotin-BCAM Ab) detection solution.

[0022] A 50 μg / μL acyl-N-hydroxysuccinimide solution was prepared using N,N-dimethylformamide as solvent and biotinyl-N-hydroxysuccinimide as solute. A 24 mg / mL rabbit anti-human BCAM polyclonal antibody solution was prepared using a 1 mol / L carbonate buffer solution (pH 9.6) as solvent and rabbit anti-human BCAM polyclonal antibody as solute. The acyl-N-hydroxysuccinimide solution and the rabbit anti-human BCAM polyclonal antibody solution were mixed at a mass ratio of 1:7. The mixture was then reacted at room temperature for 4 hours with stirring (magnetic stirrer, 200 rpm) to obtain the biotin-labeled rabbit anti-human BCAM monoclonal antibody (Biotin-BCAM). After the reaction was completed, the reaction solution containing biotin-labeled rabbit anti-human BCAM polyclonal antibody (Biotin-BCAM Ab) was placed into a dialysis bag and dialyzed at 4°C with 0.05 mol / L phosphate buffer at pH 9.2 for 12 hours, during which the dialysate was changed 3 times. Then, bovine serum albumin was added to the dialyzed reaction solution, with the amount of bovine serum albumin added limited to a concentration of 4 g / 100 mL.

[0023] The biotin-labeled rabbit anti-human BCAM polyclonal antibody (Biotin-BCAM Ab) prepared by the above process and the post-dialysis reaction solution of bovine serum albumin were mixed with glycerol at a volume ratio of 1:1 and homogenized at room temperature to form the biotin-labeled rabbit anti-human BCAM polyclonal antibody (Biotin-BCAM Ab) detection solution, which was stored at -20℃.

[0024] (3) Each kit contains 12 mL of avidin-horseradish peroxidase, 12 mL of chromogenic substrate 3',3',5,5'-tetramethylbenzidine, 1 mL of BCAM protein standard (BCAM concentration of 50 ng / mL) and 30 mL of sample diluent. The sample diluent is prepared by adding bovine serum albumin to 0.02 mol / L tris(hydroxymethyl)aminomethane buffer (Tris buffer, TBS). The amount of bovine serum albumin added is limited to a concentration of 2 g / 100 mL. The volume ratio of avidin-horseradish peroxidase to biotin-labeled rabbit anti-human BCAM polyclonal antibody detection solution is 1:1.

[0025] Example 2: Detection of BCAM level in the sample to be tested This embodiment describes the process of detecting BCAM levels in the test sample using the POP diagnostic and evaluation kit prepared in Example 1.

[0026] Source of test samples: Female subjects were selected from the outpatient, inpatient and physical examination centers of West China Second Hospital of Sichuan University. Based on existing diagnostic methods, the subjects were divided into a pelvic floor prolapse group and a healthy control group. Plasma samples were collected from both groups as test samples, totaling 70 samples. Among them, samples 1-35 were taken from the healthy control group and samples 35-70 were taken from the pelvic floor prolapse group.

[0027] Inclusion criteria for the test samples: Women aged 40–80 years, excluding those with malignant tumors, fibromyomas, pelvic masses, and mental disorders, and diagnosed with POP were included in the pelvic floor prolapse group; Women aged 40–80 years, with normal liver and kidney function and biochemical tests, no malignant tumors, normal tumor markers, no pelvic masses, no mental disorders, no pelvic floor dysfunction or history, and diagnosed without POP were included in the normal control group.

[0028] 1. Sample to be tested (1) Preparation of standard samples The BCAM protein standard provided in the kit described in Example 1 was set as the standard sample with the highest BCAM concentration and named Standard 0, with a BCAM concentration of 50 ng / mL. Then, the BCAM protein standard was diluted with the sample diluent provided in the kit described in Example 1 to form standards with BCAM concentrations of 10000 pg / mL, 4000 pg / mL, 1600 pg / mL, 640 pg / mL, 256 pg / mL, 102.4 pg / mL, and 40.96 pg / mL, respectively, and named Standard 1, Standard 2, Standard 3, Standard 4, Standard 5, Standard 6, and Standard 7, for a total of 7 standards.

[0029] (2) Sample Women from the outpatient, inpatient and physical examination centers of West China Second Hospital of Sichuan University were selected as the subjects of this study. Based on existing diagnostic methods, the subjects were divided into a pelvic floor prolapse group and a healthy control group. Anticoagulated blood samples were collected from both groups as test samples, totaling 70 samples. Among them, samples 1-35 were taken from the healthy control group and samples 36-70 were taken from the pelvic floor prolapse group.

[0030] (3) Blank control The sample diluent provided in the kit described in Example 1 is a blank control.

[0031] 2. Sample testing (1) Before testing, place all reagents and samples at room temperature (18-25℃).

[0032] (2) Add the standard, blank control and sample prepared in step 1 to the corresponding wells of the 96-well plate coated with mouse anti-human BCAM monoclonal antibody. Each standard, blank control and sample should be duplicated. The amount of sample added to each well is 100 μL. Cover the 96-well plate with a film and incubate it at room temperature for 2.5 hours under shaking conditions.

[0033] (3) After the incubation time is up, aspirate the liquid from each well and set the cleaning program on the automatic plate washer. Use 300 μL / well washing solution (0.02 mol / L phosphate buffer or 0.02 mol / L Tris buffer) to wash each well four times. Each time, the washing solution should remain in the well for 10 to 15 seconds. After cleaning, flip the 96-well plate so that the well openings are facing down and gently tap the plate on absorbent filter paper to completely remove any remaining washing solution from the wells.

[0034] (4) Add 100 μL of biotinylated antibody (BCAM Ab) detection solution to each well of the 96-well plate, then cover the 96-well plate with a film and place the 96-well plate on a shaker and incubate at room temperature for 1 hour under shaking. After the incubation time is up, aspirate the liquid from each well and clean the 96-well plate according to the method in step (3).

[0035] (5) Add 100 μL of avidin-horseradish peroxidase to each well of the 96-well plate, then cover the 96-well plate with a gel film and place the 96-well plate on a shaker and incubate at room temperature for 45 minutes under shaking. After the incubation time is up, aspirate the liquid from each well and clean the 96-well plate according to the method in step (3).

[0036] (6) Add 100 μL of 3',3',5,5'-tetramethylbenzidine to each well of the 96-well plate and incubate at room temperature in the dark for 30 minutes. After the incubation time is up, add 50 μL of stop solution to each well. The stop solution is an aqueous solution of sulfuric acid and sodium sulfite with a concentration of 2 mol / L sulfuric acid and a concentration of 0.1 mol / L sodium sulfite.

[0037] (7) Place the 96-well plate in a microplate reader and measure the A450 value (optical density or absorbance value) of each well. The results are shown in Tables 1 to 3 below. The A450 value of the blank control is 0.118.

[0038] Table 1: A450 values ​​of standard samples Table 2: A450 values ​​of samples 1-35 Table 3: A450 values ​​of samples 36-70 (8) Based on the A450 value of each standard sample and the concentration of BCAM in each standard sample, construct an A450 value-BCAM concentration standard curve (see Figure 1 The calculation formula is y = 0.0002x + 0.1616, where y is the A450 value of the sample and x is the content of BCAM in the sample.

[0039] (9) Based on the measured A450 value of each sample, the concentration of BCAM in each sample is calculated using the formula y=((max / (x-min)-1)^(-1 / Hillslope))*EC50 (see Table 6). The calculation results are shown in Table 4 and Table 5 below.

[0040] Table 4: Calculation results for samples 1-35 (BCAM concentration unit: pg / mL) Table 5: Calculation results for samples 36-70 (BCAM concentration unit: pg / mL) Table 6: Formulas and parameters for the four-parameter method The average BCAM concentration of samples 1-35 in Table 4 was calculated to be 478.95 ± 150.85 pg / mL. The average BCAM concentration of samples 36-70 in Table 5 was calculated to be 364.71 ± 84.72 pg / mL. The results show that the difference in average BCAM concentration between samples 1-35 from the healthy control group and samples 36-70 from the pelvic floor prolapse group is statistically significant.

[0041] The detection limit is set based on the ROC curve calculation results of the sample. Therefore, the detection limit for BCAM in the kit of this invention is set to 367 pg / mL, that is, a BCAM concentration below 367 pg / mL in the test sample is considered positive (i.e., female pelvic organ prolapse), and a BCAM concentration above 367 pg / mL is considered negative (i.e., no female pelvic organ prolapse).

[0042] Example 3: Specificity and Sensitivity Test This embodiment demonstrates the specificity and sensitivity of POP diagnosis and assessment using the kit described in Example 1.

[0043] (1) Specificity Source of test samples: The experimental subjects were women from the outpatient, inpatient and physical examination centers of West China Second Hospital of Sichuan University. According to the existing diagnostic methods, the experimental subjects were divided into a pelvic floor prolapse group and a healthy control group. Plasma samples were collected from both groups as test samples, totaling 70 samples. Among them, samples 1-35 were taken from the healthy control group and samples 36-70 were taken from the pelvic floor prolapse group.

[0044] The inclusion criteria and detection methods for the samples to be tested are the same as in Example 2.

[0045] The test results are shown in Table 7 below.

[0046] Table 7: Detection results of the samples to be tested Based on the ROC curve, the specificity of the kit described in this invention was calculated to be 77.14% for the test sample.

[0047] (2) Sensitivity Source of test samples: The experimental subjects were women from the outpatient, inpatient and physical examination centers of West China Second Hospital of Sichuan University. According to the existing diagnostic methods, the experimental subjects were divided into a pelvic floor prolapse group and a healthy control group. Plasma samples were collected from the two groups of women as samples, totaling 70 samples. Among them, samples 1-35 were taken from the healthy control group and samples 36-70 were taken from the pelvic floor prolapse group.

[0048] The inclusion criteria and detection methods for the samples to be tested are the same as in Example 2.

[0049] Based on the ROC curve, the sensitivity of the kit described in this invention for the test sample was calculated to be 62.86%.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. The application of BCAM in the preparation of POP diagnostic and evaluation products, characterized by: The product is a substance that uses blood samples from test subjects to detect the level of BCAM in the blood samples.

2. The application according to claim 1, characterized in that: The blood sample may be whole blood, serum, or plasma.

3. The application according to claim 1, characterized in that: The substance in question is used to detect the level of BCAM at the protein level.

4. The application according to claim 1, characterized in that: The detection methods include immunofluorescence assay, ELISA, biochip assay, and electrochemiluminescence assay.

5. The application according to claim 1, characterized in that: The products include reagent kits, test strips, or other medically feasible products.

6. A POP diagnostic and assessment product used in any one of claims 1 to 5.

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